Abstract: Mutations induced by environmental exposure to Ultraviolet (UV) light are the principle cause of most human skin cancers including melanoma. Sequencing of melanoma genomes has revealed that mutations in these samples are not evenly distributed, but instead vary heterogeneously across chromosomes. The mechanisms that underlie this mutational heterogeneity are currently unclear. We hypothesize that chromatin architecture, (i.e., the organization of DNA with DNA-bound proteins, like histones and transcription factors), alters where UV lesions form, and that the distribution of lesions in part dictates the ultimate mutation distributions seen in human cancers. To test this hypothesis, we will utilize our recently developed method to map the positions of UV lesions at single nucleotide resolution across genomes and compare the mapped locations of UV lesions to those of UV-induced mutations in yeast and human cells. In Aim I, we will analyze how genomic variations in initial UV damage formation correlate with UV-induced mutagenesis in yeast. Specifically, we will develop 6-4PP- seq to map 6-4 photoproducts at single nucleotide resolution across the yeast genome and compare the distribution of UV lesions (i.e., cyclobutane pyrimidine dimers [CPDs] and 6-4PPs) to the genome-wide mutation density in yeast following repeated UV exposures. Additionally, we will determine how genetic deficiencies in the GG-NER and TC-NER pathways influence lesion removal and mutation density. In Aim II, we will analyze the genome-wide distribution of UV- induced DNA lesions in human skin cells as a model to understand UV-induced mutagenesis in human cancers. We will optimize the CPD-seq and 6-4PP-seq methods to detect low levels of UV lesions in human cells and then utilize these methods to analyze the genome-wide distribution of UV lesions in primary human skin cell types. CPD-seq and 6-4PP-seq derived UV lesion distributions will then be compared to mutation distributions in sequenced human melanomas. Successful completion of these aims will enable us to de-convolute the influence of chromatin architecture on UV lesion formation and repair to the formation of mutations during carcinogenesis.